ABSTRACT We have identified a small molecule lead that at low doses decreases the proliferation of triple negative breast cancer (TNBC) cells while sparing normal epithelial cells. Mechanistically, our lead targets the phosphatase activity of Sts-1, a novel target that is overexpressed in TNBC and whose high expression confers poor overall survival in ER-(-) but not in ER-(+) patients. Our preliminary data show that inhibiting Sts-1 is a potent mechanism of treatment, as tumors induced by HCC1937 cell xenografts in mice carrying a Sts-1 targeting shRNA grew significantly smaller than tumors from control cells with non-targeting shRNA. In cell culture, decreasing Sts-1 expression decreased proliferation of TNBC but not of control cells. Given these activities, the goal of this research proposal is to validate the mechanism of action and to test the efficacy of our lead in an in vivo xenograft mouse model of TNBC. We will focus on TNBC because it is among the most aggressive of breast cancer subtype, with a high propensity for metastasis, poor prognosis, and short time to relapse and death. TNBC represents a clinical challenge because it does not benefit from hormonal therapies or other targeted treatments. Chemotherapy, with its adverse side effects, remains the cornerstone for TNBC treatment and chemo- agents are often administered individually or in combination; surgery and radiation are also commonly used. Our long-term goal is to develop small molecule inhibitors of the phosphatase activity of Sts-1 into a novel TNBC therapy. Our working hypothesis is that small molecule inhibitors of Sts-1 will restore c-Cbl's ability to degrade its targets (e.g. EGFR, c-Met) by ubiquitination - thereby inhibiting TNBC growth. To validate the mechanism of action of H9 and determine its potency in vivo, Aim 1 will test the effect of H9 on the activity of the c-Cbl/EGFR, c-Cbl/c-Met, and mTOR/S6K axes under acute and chronic growth conditions, investigate the cellular pathways modulated by compound H9 and compare them to Sts-1 silencing using unbiased approaches, and examine and validate binding of H9 to Sts-1 phosphatase domain using structural and biophysical techniques and site-directed mutagenesis. Aim 2 will test the ability of compound H9 to inhibit growth of TNBC tumors in vivo. We will Initiate toxicity and pharmacokinetic studies to determine compound stability and optimal dosage to achieve functional serum concentrations of compound H9 and test the ability of H9 to inhibit growth of TNBC xenograft tumors in vivo.